Cells respond to stress by increasing the synthesis of a number of molecular chaperones: cellular machines that facilitate protein folding. Heat shock proteins (Hsps) are molecular chaperones that assist general protein folding and prevent non-functional side reactions such as non-specific aggregation of misfolded or unfolded proteins, even under normal conditions. They account for 1 to 2% of total protein in unstressed cells. However, their levels of intracellular expression increase in response to protein-denaturing stressors, such as temperature change, as an evolutionarily conserved response to restore the normal protein-folding environment and to enhance cell survival. The essential chaperoning functions of Hsps are subverted during oncogenesis to make malignant transformation possible and to facilitate rapid somatic evolution.
Hsp90 (heat shock protein 90 kDa), one of the most abundant proteins expressed in cells, is a member of the heat shock protein family, up-regulated in response to stress. It has been identified as an important mediator of cancer cell survival. Hsp90 binds to a variety of target or “client” proteins, among them many steroid hormone receptors, protein kinases and transcription factors. It interacts with client-proteins by facilitating their stabilisation and activation or by directing them for proteasomal degradation. Thanks to its multifaceted ability to influence signal transduction, chromatin remodelling and epigenetic regulation, development and morphological evolution, it is considered as a promising target for cancer therapy.
The Hsp90 protein contains three well-defined domains, each of these plays a crucial role in the function of the protein. The N-terminal domain, binding site for ATP, is also the binding site for Geldanamycin, a representative of the ansamycin drugs that specifically target Hsp90. The middle domain completes the ATPase site and binds to client proteins. Finally, at the C-terminal dimerisation domain, Hsp90 forms homo-dimers where the contact sites between subunits are localised within the C-terminus in the open conformation of the dimer. During the ATPase cycle, the three domains of Hsp90 move from an ATP-free “open” state to an ATP-bound “closed” state. The N-termini also come in contact in the closed conformation of the dimer. The functions of Hsp90 include assisting in protein folding, cell signaling, and tumor repression. In unstressed cells, Hsp90 plays a number of important roles, which include assisting in folding, intracellular transport, maintenance, and degradation of proteins as well as facilitating cell signaling.
The majority of known Hsp90 inhibitors, such as the natural products belonging to the ansamycins or radicicol families or synthetic purines, bind at the ATP-site on the N-terminal domain, resulting in client protein deactivation, destabilisation and degradation. However, compounds such as novobiocin and cisplatin have been reported to bind to the C-terminal domain of Hsp90, resulting in an anti-cancer effect as well. Inhibition of Hsp90 can also be a result of inactivation through post-translational modification, typically acetylation or ubiquitinylation. When Hsp90 is inhibited, its regulatory functions are disrupted. As Hsp90 is involved in the regulation of many relevant oncoproteins, it is suggested that its inhibition will result in a broad range of biological activities, hence the Hsp chaperone molecule is an appealing target for cancer. Cancerous cells over-express a number of proteins, including PI3K and AKT and inhibition of these two proteins triggers apoptosis. As Hsp90 stabilizes the PI3K and AKT proteins, its inhibition appears to induce apoptosis through inhibition of the PI3K/AKT signaling pathway. Together with its co-chaperones, Hsp90 modulates tumour cell apoptosis, mediated through effects on AKT, tumor necrosis factor receptors (TNFR) and nuclear factor-KB (NF-κB) function. Finally Hsp90 participates in many key processes in oncogenesis such as self-sufficiency in growth signals, stabilization of mutant proteins, angiogenesis, and metastasis.
Recent studies have shown that Hsp90 also plays an important role in regulating pro-inflammatory signalling pathways. For example, agonists that stimulate NO production were reported to activate a mechanism that recruits Hsp90 to the eNOS. Interaction between Hsp90 and eNOS enhances activation of the enzyme in cells and in intact blood vessels leading to NO production. Following this discovery, Geldanamycin, a known natural inhibitor of Hsp90, was shown to be anti-inflammatory in vivo. Geldanamycin treatment was also shown to induce a significant reduction in IKK protein levels. IKK phosphorylates IκB, marking it for subsequent proteasomal degradation. It is therefore a crucial regulator of the NF-κB pathway, which holds prominent roles in inflammation and cancer. It has been shown that Hsp90 inhibitors prolong survival, reduce or abolish systemic and pulmonary inflammation, and restore normal lung function in a murine model of sepsis. Sepsis is associated with activation of pro-inflammatory mediators, including NF-κB, an important pro-inflammatory transcription factor that mediates up-regulated expression of several pro-inflammatory cytokines and chemokines, such as tumour necrosis factor α (TNF-α), IL-6, IL-8 and IL-1β, critical for amplifying the inflammatory insult. Hsp90-complexing to the glucocorticoid receptor (GR) is necessary to maintain GR in a conformation able to bind hormone. Binding of the hormone to GR causes a conformational change in the complex which results in the interaction between Hsp90 and GR to be disrupted: the receptor then translocates from the cytoplasm to the nucleus, dimerizes and binds to DNA to activate the transcription of the target genes. Hsp90 is also required for the proper functioning of several other steroid receptors, including those responsible for the binding of aldosterone, androgen, estrogen and progesterone.
HSP90 has also been implicated in a number of other conditions, such as viral infection and Alzheimer's Disease.
A group of compounds has now been identified which are potent and selective inhibitors of HSP90 and the isoforms and splice variants thereof. The compounds are characterised by the presence in the molecule of an amino acid motif or an amino acid ester motif which is hydrolysable by intracellular carboxylesterases. Compounds of the invention having lipophilic amino acid ester motifs cross the cell membrane, and are hydrolysed to the acid by said carboxylesterases. The polar hydrolysis product accumulates in the cell since it does not readily cross the cell membrane and hence the Hsp90 inhibitory activity of the compound is prolonged and enhanced. The compounds of the invention are related to the HSP90 inhibitors encompassed by the disclosures in WO2006/109075, WO2006/109085 and WO2006/117669 but differ therefrom in that the present compounds have the amino acid motif referred to above. The compounds are thus of use in medicine, for example in the treatment of a variety of proliferative disease states, where inappropriate action of HSP90 may be involved such as cancer, inflammatory and immune disorders such as rheumatoid arthritis, COPD, psoriasis, Crohn's disease, ulcerative colitis, systemic lupus erythmatosis, and disorders related to angiogenesis such as age related macular degeneration, diabetic retinopathy and endometriosis. Inhibitors of Hsp90 may be useful in the treatment of inflammation. Inflammation is mediated by a variety of soluble factors, including a group of secreted polypeptides known as cytokines. Those which mediate acute inflammation include IL-1, TNF-a, IL-6, IL-11, IL-8, G-CSF, and M-CSF. Cytokines involved in chronic inflammation can be subdivided into cytokines mediating humoral responses such as IL-4, IL-5, IL-6, IL-7, and IL-13, and those mediating cellular responses such as IL-1, IL-2, IL-3, IL-4, IL-7, IL-9, IL-10, IL-12, interferons, transforming growth factor-b, and tumor necrosisfactor a and b. Some cytokines, such as IL-1, significantly contribute to both acute and chronic inflammation. The compounds may also be of use in the protection of normal cells against the action of cytotoxic agents or in the management of viral infection or Alzheimer's Disease.